Most experiments proposed in this grant require production of AAV vector from plasmids that the individual researches design using standard recombinant DNA techniques. The goal of this (research grade) Vector Core facility is therefore the production of research grade AAV vector for pre-clinical studies in tissue culture, mice, hemophilic dogs, and other experimental systems. This function will ensure the availability. of recombinant AAV for the different projects that involve experiments with AAV vectors for expression of therapeutic transgenes. AAV vector is produced in a helper virus-free system based on large-scale plasmid transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors for the investigators. The use of a core facility will provide reproducible yield and purity of vector, and will be more cost- effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large- scale transfection of HEK-2934 cells using calcium phosphate participation, recovery and purification of recombinant AAV by cell lysis followed by column chromatography of gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will perform additional tests on purity and sterility of vector, if required for the intended experiment, and assist the researchers that requested the vectors with set up of functional assays. Standard vector preparations are expected to yield approximately vector genomes, while scale up will result in production of approximately 10/14 vector genomes per preparation. This will primarily by achieved by use of affinity column chromatography for vector purification.